Whole-mount in situ hybridization (WISH) was done according to the protocol published previously (12 (link)). Double-fluorescence in situ hybridization (dFISH) was performed adapting the WISH protocol using the TSA signal amplification system (PerkinElmer). In short, additional to the DIG-labeled ISH probe, a second fluorescein-labeled probe was generated using fluorescein labeling mix (Roche). Probe concentration was reduced to 0.02 ng/µL for both probes. Anti–fluorescein-HRP (PerkinElmer) and anti–DIG-HRP (PerkinElmer) were diluted 1:500. Signal amplification was done with amplification diluent fluorescein-tyramide and amplification diluent Cy3-tyramide (PerkinElmer) for 4 min each. Samples were mounted in Vectashield (Vector Laboratories) and pictures were taken with a Leica SP5 II confocal scanning microscope. Stacks were acquired sequentially and z-projected.
Anti dig hrp
Manufactured by PerkinElmer
Sourced in United States
Anti-DIG-HRP is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and visualize digoxigenin (DIG)-labeled molecules in various applications, such as Western blotting, ELISA, and in situ hybridization.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein tyramide, by PerkinElmer (1 mentions)
Cy3 tyramide, by PerkinElmer (1 mentions)
Anti fluorescein hrp, by PerkinElmer (1 mentions)
Sp5 2 scanning confocal microscope, by Leica (1 mentions)
Fluorescein labeling mix, by Roche (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Fluorescence microscopy, by Leica (1 mentions)
3 protocols using anti dig hrp
1
Dual-Fluorescence in situ Hybridization Protocol
2
Quantitative FISH Analysis of circATRNL1 and miR-23a-3p
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FISH was carried out for evaluation of the expression level of circATRNL1 with a Cy5-labeled probe (5′-CCACATATAATCACTCAAGCCAGAGCTGGG-3′) and of miR-23a-3p with a Cy3-labeled probe (5′-GCGGAACTTAGCCACTGTGAA-3′), respectively. Briefly, samples were fixed on glass slides with 4% paraformaldehyde. Hybridization was performed at 37°C overnight with specific probes after dehydration. Then the slides were washed in formamide/2× SSC and were incubated with anti-DIG-HRP (PerkinElmer, Boston, MA, USA) at 4°C overnight, followed by the addition of TSA fluorescent signal reaction solution (PerkinElmer), then sealed with tablets containing DAPI. The images were acquired by fluorescence microscopy (Leica, Heidelberg, Germany).
3
Circulating RNA Expression Analysis
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FISH assay was performed to evaluate the expression of circAars with a FAM-labeled probe (5’-CATCAGATTCCCTGGGATGGTTTGGC-3’) and the expression of miR-128-3p with a Cy3-labeled probe (5’-AAAGAGACCGGTTCACTGTGA-3’), respectively. In brief, BMSCs and skull paraffin sample underwent immobilization with 4% paraformaldehyde and then hybridized at 37°C overnight with specific probes. Subsequently, the samples were rinsed with 2x SSC/formamide, treated with anti-DIG-HRP (PerkinElmer, Boston, MA, USA), then treated with TSA fluorescent reagent (PerkinElmer), and then stained with 4,6-diamidino-2-phenylindole (DAPI). At final, the results were observed with CLSM (Olympus).
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